Cryo-EM structure of Pol κ−DNA−PCNA holoenzyme and implications for polymerase switching in DNA lesion bypass

Abstract

<jats:p>Replacement of the stalled replicative polymerase (Pol δ) at a DNA lesion by the error-prone DNA polymerase κ (Pol κ) restarts synthesis past the lesion to prevent genome instability. The switching from Pol δ to Pol κ is mediated by the processivity clamp PCNA but the structural basis of this mechanism is unknown. We determined the Cryo-EM structures of human Pol κ-DNA-PCNA complex and of a stalled Pol δ-DNA-PCNA complex at 3.9 and 4.7 Å resolution, respectively. In Pol κ complex, the C-terminus of the PAD domain docks the catalytic core to one PCNA protomer in an angled orientation, bending the DNA exiting Pol κ active site through PCNA. In Pol δ complex, the DNA is disengaged from the active site but is retained by the thumb domain. We present a model for polymerase switching facilitated by Pol κ recruitment to PCNA and Pol κ conformational sampling to seize the DNA from stalled Pol δ assisted by PCNA tilting.</jats:p>

Mechanisms for Maintaining Eukaryotic Replisome Progression in the Presence of DNA Damage

“…This study also revealed that PCNA can tilt up to 20°, disrupting interactions between the catalytic subunit of Pol δ and PCNA that are critical for DNA synthesis, while the polymerase remains bound to PCNA via the PIP box located in its thumb domain ( Lancey et al, 2020a ). The same group recently solved the structure of Pol κ -PCNA-DNA and stalled Pol δ -PCNA-DNA to propose a mechanism of polymerase switching ( Lancey et al, 2020b , Preprint). Therein, Pol δ releases the primer-template from the active site upon stalling, Pol κ binds to an exposed PCNA protomer in a flexible state and either actively displaces Pol δ , or Pol δ independently dissociates, from DNA to form the final Pol κ holoenzyme.…”